Nanoliposomal c-MYC-siRNA inhibits in vivo tumor growth of cisplatin-resistant ovarian cancer

ABSTRACT

The present invention discloses c-MYC-siRNA formulation as a potential therapeutic target for cisplatin-resistant ovarian cancer. It is disclosed targeting c-MYC with small interfering RNA (siRNA) in the cisplatin-resistant ovarian cancer cell line inducing a significant cell growth arrest and inhibition of cell proliferation. Apoptosis and arrest of cell cycle progression were also observed after c-MYC-siRNA-based silencing of c-MYC. Furthermore, delivering nanoliposomal c-MYC-siRNA, decreased tumor weight and number of tumor nodules compared with a liposomal-negative control siRNA.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This project is supported in part by the National Institutes of Health(NIH) 1K22CA166226-01A1 (PEVM), R25GM061838 (MGRS-RISE Program),G12MD007600 (NIMHD) and the UPR-MDACC Partnership in Cancer ResearchTraining Program.

RELATED APPLICATIONS

N/A

BACKGROUND OF THE INVENTION Discussion of the Background

Ovarian cancer presents non-specific symptoms and it has the highestmortality of all gynecological cancers. About 21,980 new ovarian cancercases and 14,270 deaths are expected in the United States in 2014.

Surgery and platinum-based adjuvant chemotherapy, such as cisplatin(CIS), are the most common treatments for ovarian cancer. Unfortunately,over 70% of women develop chemoresistance. The exact mechanism of CISresistance is not known, however, evidence indicates that activation ofthe oncogenic transcription factor c-MYC is involved in drug resistance.Our previous findings indicate that cisplatin-resistant ovarian cancercells express higher c-MYC protein levels when compared to theirsensitive counterparts.

Small modified single-stranded RNAs have emerged as a treatment modalityof exceptional promise for cancer treatment among the drugs that arecurrently under clinical trial. The mechanism by which these small RNAmolecules act are normally know as interference RNA (siRNA). Basically,small RNAs bind to messenger RNA (mRNA) and block protein synthesis.Systemic administration of these small RNA (siRNA) molecules hasremained a major challenge due to its short half-life, lack of abilityto penetrate the plasma membrane, and potential toxicity (includingactivation of the immune response). Nanoliposome-based delivery systemshave been proposed to address these concerns. Therefore, development ofsafe, easy to administer, and efficient delivery systems that achieveprolonged effect is of substantial clinical importance. Further there isa need of the incorporation of polyethylene glycol (PEG) on the surfaceof the liposomal carrier to extend blood-circulation time. Thus, aliposomal formulation containing folate on its surface will increase thestability in the circulation.

The c-myc (v-myc avian myelocytomatosis viral oncogene homolog) binds tospecific DNA sequences to activate gene expression. c-MYC regulates theexpression of genes involved in a myriad of cellular processes includingreplication, growth, metabolism, differentiation, and apoptosis.Overexpression of c-MYC has been reported in most, if not all, types ofhuman malignancies. c-MYC coordinates the activation and repression ofprotein-coding genes involved in cell growth, proliferation, loss ofdifferentiation and apoptosis; and noncoding RNAs such as microRNAs(miRNAs).

Moreover, c-MYC is commonly dysregulated in cancer, reprogramming geneexpression to facilitate cellular proliferation and tumorigenesis. Infact, the c-MYC gene is amplified in 30-60% of human ovarian cancers.Given the pivotal role of c-MYC in ovarian cancer, its therapeutictargeting in chemoresistance is important.

In conclusion, ovarian cancer is the deadliest of gynecological cancersin the United States. With fewer than 15% of cases diagnosed early,ovarian cancer continues to be characterized by late-stage presentation.Treatment for ovarian cancer usually involves surgical cytoreductionfollowed by platinum-based chemotherapy. Unfortunately, despite initial,more than 70% of ovarian cancer patients develop cisplatin resistance,relapse and therapeutic failure. Therefore, there is a need of noveltherapies focused on targets within cancer cell survival pathways foradvanced stage drug resistant such as ovarian cancer.

SUMMARY OF THE INVENTION

The small interference RNA (siRNA) is a new therapeutic modality totarget specific genes increased in cancer cells. siRNA are 22-baseribonucleic acid (RNA) molecules that bind to a specific region in thetarget gene and avoid the protein synthesis inside cell. One of themajor changes of the siRNA-based therapy is that the half-life of siRNAmolecules is very short (minutes to hours). Thus, a carrier is necessaryto encapsulate siRNA molecules and avoid their degradation in the blood.Several carries have been proposed. However, liposomes are the mostcommon drug carriers.

The major advantages of liposomes are that they are biodegradable andbiocompatible. When the size of liposomes is in the nanometer (nm) scale(1 nm is one-billionth of a meter), they are called nanoliposomes.

c-MYC is a protein highly abundant in several types of cancers. C-MYC isconsidered as an oncogene because it induces malignant transformation.We found that c-MYC is overexpressed in ovarian cancer cells that areresistant to chemotherapy. In addition, we use an internet searchabledatabase (The Cancer Genome Atlas) and found that the life expectancy ofovarian cancer patients with high c-MYC levels is lower compared withovarian cancer patients with small c-MYC levels.

Thus, we designed a nanoliposomal formulation to encapsulate c-MYC-siRNAwith therapeutic purposes.

One object of the present disclosure is to overcome the limitations ofthe previous therapies. In accordance with an exemplary embodiment thenanoliposomal formulation comprises at least a lipid, and c-MYC-siRNA,each in a ratio of 1 μg c-MYC-siRNA:10 μg DOPC(1,2-Dioleoyl-sn-glycero-3-phosphocholine), cholesterol 40% (w/w) DOPC,10% PEG-2000 (mol/mol) of DOPC.

Another object is to disclose a nanoliposomal formulation to encapsulatec-MYC-siRNA with therapeutic purposes.

Another object is to assess the molecular and therapeutic effects ofsmall-interference RNA (siRNA)-mediating c-MYC targeting incisplatin-resistant ovarian cancer.

Importantly, targeting c-MYC with small interfering RNA (siRNA) in thecisplatin-resistant ovarian cancer cell line, A2780CP20, induced asignificant cell growth arrest and inhibition of cell proliferation.This effect was corroborated in another two ovarian cancer cell lines(A2780CIS and HEYA8 ovarian cancer cells). Apoptosis and arrest of cellcycle progression were also observed after siRNA-based silencing ofc-MYC. These results were confirmed by Western blot analysis.Furthermore, in vivo delivery of c-MYC-siRNA in a murine xenograft modelof cisplatin-resistant ovarian cancer was achieved by usingDOPC/PEG-2000-based nanoliposomes. A single weekly injection ofnanoliposomal c-MYC-siRNA, during a four week period, decreased tumorweight and number of tumor nodules compared with a liposomal-negativecontrol siRNA. Finally, Liposomal c-MYC-siRNA did not induced toxic orimmune effects in mice. These data advance c-MYC-siRNA as a therapeutictarget for cisplatin-resistant ovarian cancer.

Different aspects of the invention, their configuration, and mode ofoperation will be best understood, and additional objects and advantagesthereof will become apparent, by the following detailed description of apreferred embodiment taken in conjunction with the accompanyingdrawings.

The Applicant hereby asserts, that the disclosure of the presentapplication may include more than one invention, and, in the event thatthere is more than one invention, that these inventions may bepatentable and non-obvious one with respect to the other.

Further, the purpose of the accompanying abstract is to enable the U.S.Patent and Trademark Office and the public generally, and especially thescientists, engineers, and practitioners in the art who are not familiarwith patent or legal terms or phraseology, to determine quickly from acursory inspection the nature and essence of the technical disclosure ofthe application. The abstract is neither intended to define theinvention of the application, which is measured by the claims, nor is itintended to be limiting as to the scope of the invention in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein, constitutepart of the specifications and illustrate the preferred embodiment ofthe invention.

FIG. 1 Schematic diagram of experiments.

FIG. 2 shows a schematic representation of a PEG-2000 nanoliposome.

FIG. 3 shows a Liposomal c-MYC-siRNA solution loaded in the filter.

FIG. 4 shows a serial dilution of liposomal formulations.

FIG. 5 table contains the mean size of four c-MYC-siRNA-liposomalformulation tested.

FIG. 6 is a table summarizing the size, charge and encapsulationefficiency of liposomes with different cholesterol ratios

FIG. 7 graph shows the cell toxicity results.

FIG. 8A-8B shows c-MYC expression levels in ovarian cancer cells.

FIGS. 9A-9C shows the effective c-MYC silencing in vitro, and the effectof c-MYC-siRNA-mediated c-MYC silencing on colony formation and cellproliferation.

FIGS. 10A-10C shows effect of c-MYC-siRNA-mediated c-MYC silencing oncell cycle progression and apoptosis.

FIG. 11A-11D shows the c-MYC expression in ovarian cancer human tumorswith data extracted from “the Cancer Genome Atlas” (TCGA) Data base.

FIG. 12 shows the reduction in colony formation after c-MYC-siRNA-basedsilencing of c-MYC in A2780CIS cells.

FIG. 13 Shows that in A2780 cells (very low c-MYC expression) the c-MYCsiRNA does not induce any toxic effect.

FIG. 14 shows that combination of c-MYC siRNA plus Cisplatin (CIS)further reduce tumor growth of A2780CP20 cells.

FIG. 15A-B shows the cell cycle progression proteins following c-MYCsilencing.

FIG. 16A-B shows in vitro effects of c-MYC overexpression.

c-MYC-siRNA.

FIG. 17 shows a cryo Electron micrographs indicating that thenanoliposmal formulation are small unillamelar vesicles.

FIG. 18A-18C shows the in vivo therapeutic efficacy of liposomal c-MYCsiRNA.

FIG. 19A-19D shows the effect of c-MYC-siRNA-mediated c-MYC silencing inHEYA8 ovarian cancer cells.

FIG. 20A-20D shows in vitro and in vivo characterization of liposomalformulations.

FIG. 21A-21E shows immunostimulatory effects and safety ofDOPC-PEG-c-MYC-siRNA.

FIG. 22 is a table showing the stability at room temperature ofliposomal after reconstitution.

FIG. 23 is a table showing the stability of the liposomal formulationstored at 4° C. without reconstitution.

DESCRIPTION OF THE PREFERRED EMBODIMENT

FIG. 1. Schematic diagram of experiments performed for the fullyliposomal characterization and uses. A schematic representation of thenanoliposomes is shown in FIG. 2. The nanoliposomal formulationpresented comprises lipids and c-MYC-siRNA with a preferred ratio, forexample:

1 μg c-MYC-siRNA 100:10 μg DOPC 101, cholesterol 40% 102 (w/w) DOPC, 10%PEG-2000 103 (mol/mol) of DOPC.

FIG. 2 is a representation of the nanoliposomes. Specific amounts ofthese components are mixed in excess of ter-butanol as the dissolvent.The mixture is frozen at −80° C. or lower. Then, tubes are lyophilizedto evaporate the dissolvent (ter-butanol). The lyophilized power isdissolved in DPBS. For liposomal characterization and for miceinjections liposomes are vortexed (2-5 minutes) or sonicated (10 min).

Methods

Nanoliposomal Characterization

Four c-MYC-siRNA-contained nanoliposomes formulations were prepared asdescribed above. Then, liposomes were resuspended in DPBS. The particlesize and charge were measured in a ZetaPals instrument.

Nanoliposome Size.

nanoliposomes (containing 5 μg of c-MYC-siRNA) were resuspended in 2 mlDPBS. 1.5 ml was put in a cuvette. The cuvette was put in the ZetaPalsinstrument for size measurements.

Nanoliposome Charge.

Nanoliposmes (containing 5 μg of c-MYC-siRNA) were resuspended in 2 mlDPBS. Fifty μL of this solution was mixed with 2 mL of DPBS 0.01×. Thefinal mixture was put in a cuvette, and the cuvette loaded on the ZetaPotential Analyzer machine.

Encapsulation Efficiency.

1. 20 μg c-MYC-siRNA-containing liposomes were dissolved in 400 μL ofDPBS. Four-hundred μL of the mixture was loaded in a centrifugal filterF of 50 kDa (Millipore). The filter F was centrifuged at 7,500 rpm for10 min. 10 μL of the liquid on the bottom of the tube T was used tocalculate the c-MYC-siRNA concentration with the NanoDrop-1000instrument. Naked c-MYC-siRNA dissolved in DPBS (no liposomes) was usedas a control. Because the naked c-MYC-siRNA is completely dissolved allamount should go through the filter F after centrifugation. 400 μL oftritox X-100 (0.5%) was added in the top of the filter and the tube(containing the filter) was centrifuged again.

The encapsulation efficiency was calculated with the following equation:% E=(total siRNA−free siRNA/total siRNA)×100

As shown in FIG. 3 the Liposomal c-MYC-siRNA solution is loaded in thetube. The tube is centrifuged. The non-encapsulated NS c-MYC-siRNA crossthe filter F. The encapsulated c-MYC-siRNA ES remains in the filter F.

Liposomal Toxicity.

For toxicity; liposomal were prepared without c-MYC-siRNA. 50 μg ofDOPC-containing nanoliposomes were dissolved in 2 ml of sterile DPBS.Serial dilutions of nanaliposomes were prepared in RPMI (10% fetalbovine serum) (see FIG. 4). One-hundred μl each dilution was added tothe human ovarian cancer cell line, A2780CP20. Cells were incubated72-hr with the liposomes. Then, the cell survival was calculated withthe AlamarBlue dye (4). The percentage of survival was calculatedrelative to cells without any treatment.

The present disclosure includes in vitro and in vivo studies.

Nanoliposomal Size and Charge.

The FIGS. 5 and 6 contains the mean size of four siRNA-liposomalformulation tested.

The following amount are in μl.

c-MYC-siRNA concentration stock=1 mg/ml

FIGS. 5 and 6 disclose several concentration combinations of theformulation in accordance with principles of the present disclosure.According to the results the selected liposomal formulation for in vitroand in vivo studies is the number 2 in FIG. 5 (table). 50 μg of DOPCliposomes were resuspended in 2 ml of DPBS for in vitro experiments.

Particle Charge and Encapsulation Efficiency

FIG. 6 shows a table summarizing the size, charge and encapsulationefficiency. The charge was measured for formulations 2 (see FIG. 5) atdifferent cholesterol ratios. FIG. 6 indicate that the formulationcontaining 50% or 25% cholesterol are smaller in size and are the objectof the present disclosure. The encapsulation efficiency shows thatliposomal formulations containing 25% and 50% cholesterol were aroundthe same. Thus, the formulation 2 was used for in vivo experimentation.

Toxicity.

The graph, as shown in FIG. 7, shows that any of the dilutions of theformulation number 2 induced toxicity in the cells, which demonstratethe safety of this liposomal formulation obtained from the toxicityexperiments. As shown in FIG. 7, regarding the cell toxicity results,the only dilution that reduced cell proliferation was the higherdilution. This reduced cell proliferation in 20%. However, this dilutioncontains much more liposomes that the ones injected in mice.

c-MYC expression levels in ovarian cancer cells is shown in FIG. 8.Ovarian cancer cell lines that are more resistant to chemotherapyexpress higher levels of c-MYC compared with cells that are moresensitive to chemotherapy. A densitometric analysis of the bands in theFIG. 8A confirmed these results (FIG. 8B). FIG. 9A-9C shows the effectof c-MYC-siRNA-mediated c-MYC silencing on colony formation and cellproliferation. As shown in FIG. 9A, A2780CP20 cells were transfectedwith 200 nM of two different c-MYC-siRNA targeting c-MYC reducedsignificantly the c-MYC levels compared with the control-siRNA orC-siRNA. As shown in FIG. 9B, colonies ≧50 were scored under a lightmicroscope. The % of clonogenicity was calculated relative to C-siRNA(100 nM). The c-MYC-siRNA reduced in more than 50% the number ofcolonies compared with the control. As shown in FIG. 9C, A2780CP20 cellswere transfected with a serial dilution of C-siRNA or c-MYC-targetedsiRNAs. Cell proliferation was calculated spectrophotometrically usingthe Alamar blue dye. Averages±SEM are shown for at least threeindependent experiments. Significant difference at P<0.001 (***) ispresented based on Student's t-test. C-MYC-siRNA reduced significantlythe cell growth of cisplatin resistant cells.

FIG. 10A-10C shows the effect of c-MYC-siRNA-mediated c-MYC silencing oncell cycle progression and apoptosis. A2780CP20 cells were transfectedwith 200 nM C-siRNA and c-MYC-targeted siRNA. As shown in FIG. 10A, cellcycle progression was evaluated using propidium iodide (PI) afterforty-eight hours of c-MYC-siRNA transfection. As shown in FIG. 10B,apoptosis was evaluated using FITC-Annexin V/PI after seventy-two hoursc-MYC-siRNA transfection. Averages±SEM are shown for at least twoindependent experiments. Significant difference, relative to C-siRNA, atP<0.01 (**) and P<0.0001 (****) are presented based on ANOVA andStudent's t-test. The c-MYC-siRNA activated apoptosis in almost 30%compared with the control siRNA. FIG. 10C is representative Westernblots show changes in the levels of key proteins involved in cell cycleprogression and apoptosis following c-MYC-siRNA transfection.

FIG. 11A-11D shows Kaplan-Meier survival curves indicating that ovariancancer patient with higher c-MYC expression levels live less thanpatients with lower c-MYC expression levels. Transfection of c-MYC-siRNAalso reduced the cell proliferation of another cisplatin (CIS) resistantovarian cancer cells (A2780CIS) (FIG. 12). In addition, transfection ofc-MYC siRNA in cells with very low c-MYC expression (A2780, FIG. 8A) didnot induce cell toxicity (FIG. 13). Furthermore, the combination ofc-MYC siRNA and cisplatin further potentiated the c-MYC siRNA effect(FIG. 14). Slicing of c-MYC also induced changes in proteins involved Icell cycle progression (FIG. 15A-15B).

c-MYC was overexpressed in cells with low c-MYC levels (A2780)(FIG.16A). The rate of cell proliferation of these cells were higher comparedwith empty vectors (FIG. 16B). The effect of nanoliposomal-c-MYC-siRNAwas tested on target depletion, and therapy efficacy. Nude mice wereinjected intraperitoneally (i.p.) with A2780CP20 cells. A nudemice-bearing tumors were injected i.p. with nanoliposomal siRNAs. Sevendays post-injection, mice were sacrificed and the tumors were dissected.As shown is FIG. 18A, the anti-tumor effects ofnanoliposomal-c-MYC-siRNA as compared to C-siRNA were tested intumor-bearing mice. Tumor weight, as shown in FIG. 18B and number ofnodules, as shown in FIG. 18C, were assessed in five treatment groups(10 mice each). All nanoliposomal-c-MYC-siRNAs and CIS treatments wereinjected i.p. once a week for 4 weeks. In vitro experiments wererepeated in HEYA8 ovarian cancer cells which express high c-MYC levels(FIG. 19A-19D). Additional liposomal c-MYc-siRNA formulation wasperformed (FIG. 20A-20D) and FIGS. 22 and 23). The safety ofnanoliposmes were tested in mice (FIG. 21A-21E).

Results

-   -   Western blot analysis was used to measure the c-MYC protein        levels in a panel of ovarian cancer cells. Cisplatin-resistant        cells expressed higher levels of c-MYC protein when compared to        their sensitive counterparts.    -   c-MYC targeting by small interference RNA (siRNA) in        cisplatin-resistant ovarian cancer cells induced a significant        cell growth arrest and inhibition of cell proliferation.    -   Apoptosis and arrest of cell cycle progression were also        observed after siRNA-based silencing of c-MYC. Results were        confirmed by Western blot analysis.    -   Nanoliposomal c-MYC-siRNA reduced the expression of c-MYC        protein compared to the nanoliposomal C-siRNA group in mice        bearing tumors.    -   Cisplatin treatment by itself did not induce a significant        effect on tumor growth. On the other hand, decreased tumor        weight was observed in the nanoliposomal c-MYC-siRNA group        compared to the nanoliposomal C-siRNA group. This effect was        further potentiated by cisplatin treatment (c-MYC-siRNA vs.        c-MYC-siRNA+CIS).    -   Nanoliposomal c-MYC-siRNA induced a decrease in the number of        tumor nodules compared to the nanoliposomal C-siRNA group.        However, no further reduction in the number of tumor nodules was        observed when cisplatin was combined with nanoliposomal        c-MYC-siRNA.    -   These data advance c-MYC as a therapeutic target for        cisplatin-resistant ovarian cancer.

Expression of c-MYC in Human Ovarian Cancer Patients and Ovarian CancerCells

To determine the clinical relevance of c-MYC in drug resistant ovariancancer, the c-MYC mRNA levels were correlated with clinical data fromovarian cancer patients. Ovarian cancer patient data were downloaded andanalyzed from “TCCA”. Level 3 Illumina RNASeq “gene.quantification”files were used to extract MYC expression. Statistical analysis of c-MYCmRNA expression and clinical data from patients with high grade serousovarian cancer showed that the PFS as shown in FIG. 11A through FIG. 11Band the OS as shown in FIG. 11C through FIG. 11D were significantlyreduced for patients with higher c-MYC expression levels.

As shown in FIG. 11A through 11F, the entire cohort was separated intotwo sets, the training set (219 patients) shown in FIG. 11A and thevalidation set (110 patients) shown in FIG. 11B. The log-rank testrevealed that the recurrence of the disease (expressed as percentagedisease free) occurred significantly (P=0.0277) faster for patients withhigher c-MYC expression levels as shown in FIG. 11A. This findings werecorroborated with further analysis with the validation set cohort(P=0.0289) as shown in FIG. 11B. In addition, the overall survival(expressed as the percentage survival) was significantly reduced forpatients with higher c-MYC expression values (P=0.0058) as shown in FIG.11C. Statistical analysis with the validation set cohort corroboratedthese findings (P=0.0138).

To assess c-MYC protein levels, a panel of multiple ovarian cancer celllines was evaluated by Western blot analysis. Interestingly,cisplatin-resistant cells (A2780CP20 and A2780CIS) expressed higherlevels of c-MYC protein when compared to their sensitive counterparts(A2780) as shown in FIG. 8A-8B. The densitometric analysis of the bandintensities confirmed these findings. The taxane-resistant ovariancancer cells SKOV3.TR and HEYA8.MDR exhibited similar c-MYC levels whencompared to their taxane-sensitive counterparts SKOV3ip1 and HEYA8,respectively. Together, these results show that c-MYC is a clinicalrelevant target for cisplatin resistant ovarian cancer patients.

Effects of c-MYC Silencing on Cell Growth, Proliferation, Apoptosis andCell Cycle Progression.

FIG. 9A through 9C are directed to siRNA-based silencing of c-MYC. Twodifferent siRNAs targeting exon 2 and exon 3 of the human c-MYC sequence(NM_002467) were used. FIG. 9A shows A2780CP20 cells (2×10⁵) transfectedwith 200 nM c-MYC-siRNA. Total protein was isolated fromsiRNA-transfected cells for Western blot analysis. Densitometricanalysis of the intensities of the bands was calculated relative to theC-siRNA. Averages±SEM are shown (****P<0.0001). FIG. 9B shows A2780CP20cells (6×10⁴) seeded into 6-well plates and 24-hr later 100 nMc-MYC-siRNA (2) or 100 nM C-siRNA was added to the cells. Eight hourspost-transfection, 1000 cells were seeded into 10-cm Petri dishes. Sevendays later, cells were stained and colonies of at least 50 cells werescored under a light microscope. The % of clonogenicity was calculatedrelative to C-siRNA. Averages±SEM are shown for three independentexperiments (***P<0.001). FIG. 9C shows A2780CP20 cells (2×10³) seededinto 96-well plates and 24-hr later cells were transfected with a serialdilution of C-siRNA or c-MYC-targeted siRNAs. Cell viability wascalculated 72-hr post-transfection. Percentages were obtained afterblank OD subtraction, taking the untreated cells values as anormalization control. Averages±SEM are shown.

FIG. 10A-10C shows the effect of c-MYC-siRNA-mediated c-MYC silencing oncell cycle progression and apoptosis. A2780CP20 cells were transfectedwith 200 nM C-siRNA or c-MYC-targeted siRNA. The c-MYC siRNA activatedapoptosis in almost 30% compared with the control siRNA. FIG. 10C isrepresentative Western blots show changes in the levels of key proteinsinvolved in apoptosis following c-MYC siRNA transfection.

FIG. 12 shows A2780CIS cells (9×10⁴) seeded into 6-well plates and 24-hrlater 100 nM c-MYC-siRNA (2) or 100 nM C-siRNA was added to the cells.Eight hours post-transfection, 2500 cells were seeded into 10-cm Petridishes. Ten days later, cells were stained and colonies of at least 50cells were scored under a light microscope. c-MYC siRNA reduced cellproliferation of A2780CIS cells in more than 50% compared with thecontrol siRNA (****P<0.0001).

c-MYC siRNA did not induce cell toxicity in cells with low c-MYCexpression levels (FIG. 13). In addition, the c-MYC-siRNA effect ispotentiated by c-MYC (FIG. 14). C-MYC siRNA also had effects in proteinsrelated with cell cycle progression (FIG. 15) AS shown in FIG. 8Athrough FIG. 15, it is clearly disclosed the biological effects of c-MYCsilencing in cisplatin resistant ovarian cancer cells. Western blotanalysis confirmed that the two siRNAs against c-MYC reduceddramatically the levels of c-MYC in the cisplatin-resistant ovariancancer cell lines. Similar results were observed when c-MYC-siRNA wastransfected into HEYA8 ovarian cancer cells (further see FIG. 19A-19D).Dose-dependent inhibition of cell growth was observed after 72-hr ofc-MYC-targeted siRNAs treatment. The c-MYC siRNA growth inhibitoryeffects were observed even at doses as low as 12.5 nM of c-MYC-targetedsiRNAs. Treatment with c-MYC-targeted siRNA also induced long-term incell proliferation as evidenced in colony formation assays. Transienttransfection of c-MYC-siRNA (2) in A2780CP20 cells reduced significantly(55%, **p<0.001) the number of colonies formed after 7 days in culturecompared with the c-siRNA-transfected cells. Similarly, transfection ofc-MYC-siRNA (2) in A2780CIS and HEYA8 cells significantly reduced (48%and 70% reduction, ****P<0.0001 and ***P<0.001, respectively) the numberof colonies formed after 10 days in culture (FIG. 9B and FIG. 19B). Onthe other hand, silencing c-MYC in A2780 cisplatin-sensitive ovariancancer cells, which express low c-MYC levels, induced negligible changesin cell proliferation (FIG. 13). Combination of a low-active c-MYC-siRNA(2) dose with a relatively low cisplatin dose (2 μM) induced significant(*P<0.05) additive-like effects in cell growth inhibition of A2780CP20cells (FIG. 14) compared to c-MYC-siRNA (2) alone. These data suggestthat c-MYC levels are associated with the sensitivity of ovarian cancercells to cisplatin treatment.

Effect of c-MYC Overexpression in the Sensitivity of Ovarian CancerCells to Cisplatin Treatment

As shown in FIG. 16A-16B the directed effect effects of c-MYCoverexpression in ovarian cancer cells. A2780 cells (9×10⁴) were stablytransfected with an empty vector (EV) or with a c-MYC-containing vector.FIG. 16A shows a Western blot analysis performed as previouslydescribed. Compared with untransfected cells or with empty vectorclones, the c-MYC overexpressing clones showed higher c-MYC proteinlevels. FIG. 16B shows stable transfected A2780 cells exposed to CIS (1μM final concentration)-containing RPMI-1640 media. Cell viability wascalculated. Averages±SEM are shown relative to A2780-EV1 (***P<0.001,****P<0.0001) or to A2780-EV2 (###P<0.001, ####P<0.0001). FIGS. 18A, 18Band 18C are the effects of targeting c-MYC in ovarian mouse models withnanoliposomal formulations. liposomal-siRNAs were reconstituted in Ca²⁺and Mg²⁺-free PBS. A Cryo-electron micrograph is shown in FIG. 17,discloses that the majority of the particles are small unilamellarvesicles in the 100-150 nm range. The hole in the image is afenestration in the carbon support, which measures 1.2 microns indiameter. Nude mice were injected i.p. with A2780CP20 cells and randomlyallocated in the groups FIG. 18A shows a Western blot analysisdisclosing that c-MYC-siRNA-DOPC-PEG treatment reduced c-MYC proteinlevels in vivo. Therapy began one (1) week after tumor cell inoculation.FIG. 15B shows mean tumor weight and FIG. 15C shows the number ofnodules recorded after 4-weeks. Averages±SEM are shown (*P<0.05,****P<0.0001).

These data suggest that c-MYC contributes to the cisplatin resistantphenotype of ovarian cancer cells.

Characterization of Liposome-siRNA Formulations

Dynamic light scattering showed that the liposomes used in this studywere slightly negative, and around 100-150 nm in diameter (See FIG. 5,FIG. 6 and FIG. 17). The percentage of cholesterol induced changes inthe size but not in the surface charge (zeta potential) of the liposomalformulations (See FIG. 6). The efficiency of c-MYC-siRNA encapsulationwas slightly higher for liposomes with 50% cholesterol (w/w DOPC) ascompared with liposomes with 25% cholesterol (w/w DOPC) (See FIG. 6). Acryo-EM micrograph confirmed the particle size (100-150 nm) and showedthat the majority of the liposomes are small unilamellar vesicles (seeFIG. 17). The kinetics of c-MYC-siRNA release from liposomes with 25%cholesterol was slower in the first hours compared with liposomes with50% cholesterol (see FIG. 20A). However, the kinetics of c-MYC-siRNArelease was constant over time for liposomes with 50% cholesterolcompared with liposomes with 25% cholesterol. For these reasons,liposomes with 50% cholesterol were used for further studies. Theability of the liposomes to protect the stability of the c-MYC-siRNAfrom serum nucleases was evaluated in vitro. Results showed thatc-MYC-siRNA degradation occurred faster for naked-siRNA compared toliposomal-siRNA (see FIG. 20B). Furthermore, the liposomal-c-MYC-siRNAformulation was not toxic in vitro even at DOPC concentrations as highas 50 μM. (see FIG. 20C). The size and charge of the reconstitutedliposomal-c-MYC-siRNA formulations were stable over 2-hr at RT (See FIG.22) and 4-weeks at 4° C. (FIG. 23). In vivo studies showed that a singleinjection of empty liposomes or liposomal-c-MYC-siRNA formulations didnot induce early (5-hr) or late (24-hr) immune response (See FIG.21A-21B). Repeated doses of liposomal-c-MYC-siRNA formulations (a singleinjection per day for 4 days, during 3 weeks) were not toxic for mice asindicated by the LDH activity or urea levels (see FIG. 21C-21D), whichwere similar to the control group (saline solution, only). No weightloss was noted during the treatment period (See FIG. 21E).

Therapeutic Effect of PEG-Liposomal c-MYC-siRNAs

DOPC-PEG-cholesterol-based nanoliposomes were used for in vivoc-MYC-siRNA delivery. First, we assessed whether the c-MYC silencing invivo. Nude mice-bearing A2780CP20 tumors were injected i.p. with 5 μg ofPEG-liposomal-c-siRNA or 5 μg of PEG-liposomal-c-MYC-siRNA. Seven dayspost-injection, mice were sacrificed and the tumors were dissected.c-MYC-siRNA-liposomal reduced the expression of total c-MYC at sevendays post-injection (see FIG. 15D) compared with the control groups. Theanti-tumor effects of c-MYC-siRNA as compared to c-siRNA were tested inA2780CP20 tumor-bearing mice. Tumor weight and number of nodules wereassessed in five treatment groups (10 mice each): (a) C-siRNA, (b)cisplatin (CIS) alone, (c) c-MYC-siRNA, (d) C-siRNA plus CIS, and (e)c-MYC-siRNA plus CIS. All siRNA-DOPC (5 μg siRNA/injection) and CIS (160μg/injection) treatments were injected i.p. once a week for 4 weeks. CIStreatment by itself did not induce a significant effect on tumor growth.On the other hand, decreased tumor weight was observed in thec-MYC-siRNA group (*p<0.05) compared with c-siRNA group of animals. Thiseffect was further potentiated by CIS (c-MYC-siRNA vs. c-MYC-siRNA+CIS)(***P<0.001). c-MYC-siRNA induced a decrease in the number of tumornodules (*p<0.05) compared with c-siRNA group (see FIG. 15F).

In the present disclosure, it was shown that high levels of c-MYC areassociated with faster recurrence and poor overall survival of patientswith high grade serous ovarian cancer, and with cisplatin resistance inovarian cancer cells. Another object of the present disclosure was toprovide that c-MYC-siRNA-based silencing of c-MYC inhibits cellproliferation in vitro and reduces tumor growth in xenograft models ofcisplatin-resistant ovarian cancer. c-MYC, an oncoprotein highlyabundant in several types of cancer, is considered an undruggablemolecule by virtue of its flat protein surface. Thus, the evidence wepresent here shows that siRNA-based c-MYC targeting is a therapeuticmodality for ovarian cancer patients expressing high c-MYC levels,including those that are resistant to cisplatin treatment. The c-MYCtranscription factor, which regulates approximately 15% of all humangenes, plays an important role in a myriad of biological processesincluding cell growth and proliferation, cell cycle progression,apoptosis, angiogenesis, senescence and genomic instability. Inaddition, c-MYC regulates the expression of not only a particular groupof genes but acts in concert with RNA polymerase and transcriptionfactors as a universal amplifier of gene expression in embryonic stemcells and tumor cells. In fact, c-MYC amplification has been reported inmultiple malignancies including ovarian cancer. In other tumor types,c-MYC expression levels have been associated with drug resistance. Forinstance, Sakamuro and co-workers have shown that c-MYC oncoproteinincreases cisplatin resistance by decreasing production of the c-MYCinhibitor bridging integrator 1 (BIN1). The present disclosure relatesto the role of ectopic expression of c-MYC in decreasing the sensitivityof ovarian cancer cells to cisplatin treatment.

Current adjuvant chemotherapy for ovarian cancer includes cisplatin andpaclitaxel; unfortunately, the majority of the patients developchemoresistance which leads to therapeutic failure. Thus, the presentdisclosure provides further evidence that c-MYC is a plausible targetfor ovarian cancer patients with high c-MYC expression levels. Moreover,the findings that the c-MYC-targeted siRNA did not affect the viabilityof cells with low c-MYC protein levels, suggests that c-MYC could beconsidered as a potential biomarker and an indicative of chemotherapyresponse.

We have shown that c-MYC siRNA-based silencing induces short- andlong-term effects in cell growth and proliferation. These effects wereassociated with both apoptosis induction, and cell cycle arrest. Futurestudies should determine the c-MYC-regulated anti-apoptotic genesassociated with the cisplatin resistance in ovarian cancer cells.Further one of the major cell cycle inhibitory proteins, p27, wasincreased following c-MYC depletion. Similarly, decreased levels of CDK4and cyclin D3 following c-MYC silencing occurred by the ability of c-MYCto transcriptionally regulate the expression of these proteins.

In conclusion, the present formulation and method for DOPC-PEG-liposomalc-MYC-targeted siRNA alone or in combination with chemotherapy isefficacious against ovarian cancer.

The invention is not limited to the precise configuration describedabove. While the invention has been described as having a preferreddesign, it is understood that many changes, modifications, variationsand other uses and applications of the subject invention will, however,become apparent to those skilled in the art without materially departingfrom the novel teachings and advantages of this invention afterconsidering this specification together with the accompanying drawings.Accordingly, all such changes, modifications, variations and other usesand applications which do not depart from the spirit and scope of theinvention are deemed to be covered by this invention as defined in thefollowing claims and their legal equivalents. In the claims,means-plus-function clauses, if any, are intended to cover thestructures described herein as performing the recited function and notonly structural equivalents but also equivalent structures.

All of the patents, patent applications, and publications recitedherein, and in the Declaration attached hereto, if any, are herebyincorporated by reference as if set forth in their entirety herein. All,or substantially all, the components disclosed in such patents may beused in the embodiments of the present invention, as well as equivalentsthereof. The details in the patents, patent applications, andpublications incorporated by reference herein may be considered to beincorporable at applicant's option, into the claims during prosecutionas further limitations in the claims to patently distinguish any amendedclaims from any applied prior art.

What is claimed is:
 1. A compound for treating cancer cells in a mammal,wherein said compound is a nanoliposomal formulation comprising a ratioof 1 μg c-MYC-siRNA, 10 μg 1,2-Dioleoyl-sn-glycero-3-phosphocholine(DOPC), cholesterol in the range between 25% to 50% (w/w) of DOPC and10% DSPE-PEG-2000 (mol/mol) of DOPC, wherein the nanoliposomalformulation has a mean particle size in the range of 93 to 116.2nanometers; and wherein the nanoliposomal formulation has a mean Zpotential in the range of −2.50 to −2.81 millivolts.
 2. The compound ofclaim 1, wherein the selected concentration for said nanoliposomalformulation comprises cholesterol 40% (w/w) of DOPC.